Thermo-sensitive, mucoadhesive or dermoadhesive, and penetration-enhancing formulations for topical delivery of therapeutics

ABSTRACT

The present invention provides thermo-sensitive, mucoadhesive biopolymer formulations that enhance the penetration of therapeutics across the skin or mucosal surfaces. In a preferred embodiment, the biopolymer formulation comprises co-polymer of poloxamer 188 and propylene glycol, laurocapram and, optionally, one or more therapeutic agents. Also provided are uses of the biopolymer formulations for topical therapy of cancer including cervical cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/008,004, filedSep. 27, 2013, now U.S. Pat. No. 9,056,137, which is the U.S. nationalstage application of International Patent Application No.PCT/US2012/031084, filed Mar. 29, 2012, which claims the benefit of U.S.Provisional Patent Applications Ser. No. 61/470,551, filed Apr. 1, 2011,and Ser. No. 61/602,769, filed Feb. 24, 2012 the disclosures of whichare hereby incorporated by reference in their entireties, including allfigures, tables and nucleic acid sequences.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Mar. 28, 2012 and is 1 KB. The entire contents ofthe sequence listing is incorporated herein by reference in itsentirety.

BACKGROUND

Cervical cancer is a fatal disease if not detected and treated early.99.7% of cervical cancer cases are etiologically associated with atleast one of 15-18 oncogenic types of human papillomavirus (4-6). Over85% of cervical cancer occurs in developing countries and otherhistorically underserved low-resource populations where it is theleading cause of death from cancer among women (1). Most high-gradecervical cancer can be prevented if pre-cancerous lesions or early-stagecervical cancer becomes diagnosed and treated.

At present, pre-cancerous lesions or early-stage cervical cancer can bedetected using inexpensive screening methods, such as visual inspectionof the cervix using acetic acid (vinegar) or Lugol's solution (iodine),or by using a new and affordable HPV-DNA test (careHPV, Qiagen) that candetect 14 high-risk types of HPV. However, there is a lack of affordabletherapies for pre-cancerous cervical dysplasia. The costly and invasivetherapies currently in use (cryotherapy, cone biopsies and loopelectrosurgical excision procedure) all require both an establishedclinical infrastructure as well as highly trained medical personneltypically in the form of a dedicated gynecologic oncology service. Suchresources are unavailable to large high-risk populations who haveneither the economic means nor transportation options to access suchclinical services. Thus, while limitations to screening in low resourcesettings appear to have been adequately addressed, there remains anunmet and urgent global need for an alternative therapy for those whohave positive test results. The remaining challenge is to create a noveltherapy for pre-cancerous cervical dysplastic lesions caused byhigh-risk types of HPV and prevent their progression to carcinoma insitu and/or invasive cervical cancer. Such a therapy should beinexpensive, easily self-administered or readily applied with assistanceby a trained healthcare worker in a local clinic, and be effective as asingle dose therapy that can be used in a single visit “screen, treatand prevent” public healthcare model that can be implemented even in themost impoverished populations where extremes of temperature may exist,space is limited and running water and electricity are unavailable.

BRIEF SUMMARY

The present invention provides novel co-polymer and drugpenetration-enhancer formulations as a non-invasive delivery system fordirect topical delivery of one or more chemotherapeutic agents for thetreatment of cervical dysplasia aimed at preventing progression tocarcinoma in situ and/or invasive cervical cancer. Advantageously, theco-polymer/enhancer formulations of the present invention arethermo-sensitive, mucoadhesive or dermoadhesive, and enhance thepenetration of small and large therapeutically active compounds such asproteins greater than 52 amino acids across normal or diseased skin(transdermal delivery) or mucosa (transmucosal delivery). Theseproperties facilitate the delivery of stable and active compoundscontained in such co-polymer/enhancer formulations with properties thatallow them to be solid phase at room temperature for ease of insertionand directed application to the cervical transformation zone, instantlymelt at physiological temperature, adhere to mucosa and enhance thepenetration of a chemotherapeutic drug across the entire dysplasticsquamous epithelium. In a preferred embodiment, the co-polymer/enhancerformulations comprise a mixture of poloxamer 188 (P188), propyleneglycol (PG) and laurocapram (LP) that can be customized and optimizedfor the delivery of one or more therapeutic agents or compounds.

In addition, the present invention provides uses of co-polymer/enhancerformulations for thermo-sensitive, dermoadhesive or mucoadhesive andpenetration-enhanced topical delivery of a broad range of compounds totreat both neoplastic and non-neoplastic proliferative lesions of theskin or mucosal surfaces. In certain embodiments, theco-polymer/enhancer formulation of the present invention enhancesdelivery of therapeutic agents across normal or diseased skin or mucosalsurfaces including, but not limited to, cervix, vagina, vulva, anus,rectum, eye, ear, oral cavity, nasopharynx, larynx, and head and neckfor topical therapy.

In one embodiment, the present invention can be used for treatment ofneoplastic and non-neoplastic proliferative diseases including, but notlimited to, HPV-associated cervical lesions such as pre-cancerouslow-grade cervical dysplasia including cervical intraepithelialneoplasia stage I or II (CIN I, CIN II) and high-grade dysplasia (CINIII); vulvar cancer; vaginal cancer or tumor; endometrial cancer;laryngeal carcinoma; head and neck cancer; skin cancer; skin tags;common HPV-associated warts of the hands, feet and extremities; andpsoriasis.

In one embodiment, the present invention may be used as a transdermal ortransmucosal deliver system (or device) for small and large therapeuticcompounds and biomolecules into subcutaneous or submucosal tissue, orinto the systemic circulation as an alternative to subcutaneousinjections with needles or intravenous delivery with indwellingcatheters.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a thermal profile of the *Br1-containing P188/PG/LPco-polymer/enhancer formulation. Differential Scanning calorimetrycurves show the thermal property of the drug delivery formulation, whichcontains 1 mM *Br1, a 38 amino acid cytotoxic peptide, in variousproportions of poloxamer 188 (P188) and propylene glycol (PG) with theaddition of 0.4 M laurocapram (LP).

FIGS. 2A, 2B and 2B′ show micrographs of mouse cervical squamousepithelium (original magnification 10×). (A) H&E stain of squamousepithelium lining the cervical canal and its left and right branches.The squamo-columnar junction is not shown in this section. (B)Fluorescence micrograph of a consecutive section, showing that theP188/PG-containing drug delivery formulation melts and adheres to thecervical mucosal surface in both cervical/uterine horns. (B′)Enlargement of detail of box in panel B, showing a gradient oflaurocapram-enhanced penetration 6 hours after insertion of thepeptide-containing drug delivery formulation across the thicknessdelineated by the arrow in the drug delivery formulation containingP188, PG and LP (left cervical canal), but not in the absence of LP(right cervical canal). Intense staining of mucoadherent FITC-conjugated*Br1 is observed on the mucosal surface of both cervical canals anduterine horns. Intense staining shows that FITC-conjugated *Br1 meltsand adheres to the mucosal surface of the cervical transformation zonespanning the endocervical canal and endocervix leading to both uterinehorns.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is an amino acid sequence of a TRIP-Br1 decoy peptide(*Br1).

SEQ ID NO: 2 is an amino acid sequence of a TRIP-Br2 decoy peptide(*Br2).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides co-polymer/enhancer formulations fortopical and transdermal or transmucosal delivery of compounds.Advantageously, the co-polymer/enhancer formulations of the presentinvention are thermo-sensitive, mucoadhesive or dermoadhesive, andenhance the penetration of therapeutics across skin or mucosal surfaces.Also provided are therapeutic uses of the co-polymer/enhancerco-polymer/enhancer formulations for topical treatment of cancer,including cervical cancer.

In a preferred embodiment, the delivery system comprises co-polymers ofpoloxamer 188 (P188) and propylene glycol, the penetration-enhancerlaurocapram and, optionally, one or more therapeutic agents. In apreferred embodiment, the delivery formulation of the present inventioncan be used for topical delivery of large cytotoxic peptides, such asTRIP-Br decoy peptides including the TRIP-Br1 decoy peptide (*Br1)and/or the TRIP-Br2 decoy peptide (*Br2). TRIP-Br decoy peptides targetdisruption of the TRIP-Br integrator function at E2F-responsivepromoters, which induces a novel mechanism of cell death inproliferating cells (8-10); therefore, topical delivery of the TRIP-BRdecoy peptides according to the present invention not only provideslocal therapy of pre-cancerous cervical dysplasia, but also preventsprogression of pre-cancerous conditions to cervical cancer.

As demonstrated in the Example, the co-polymer/enhancer formulations ofthe present invention are thermo-sensitive; that is, solid at roomtemperature for ease of insertion into the cervical transformation zoneand instantly melt at physiological temperature. Furthermore, theco-polymer/enhancer formulations adhere to and enhance peptidepenetration across cervical mucosa.

In one embodiment, the present invention can be used for topical therapyof human HPV-associated low-grade cervical dysplasia definedcytologically as cervical intraepithelial neoplasia stage I or II (CINI, CIN II) or for high-grade dysplasia (CIN III) to prevent progressionto carcinoma in situ (CIS) and invasive cervical cancer (11).

Advantageously, the co-polymer/enhancer formulations of the presentinvention allow for non-invasive, targeted delivery of therapeuticsacross the skin and mucosal surfaces. Additionally, the presentco-polymer/enhancer formulations use safe, inexpensive ingredients, areeasy to administer and are suitable for use in a wide range of clinicalsettings. The co-polymer/enhancer formulations can easily beadministered by healthcare workers or by self-administration by thepatient, and under conditions of extreme temperature, high humidity,poor lighting, lack of space or lack of adequate supply of electricityor water.

Co-Polymer/Enhancer Formulation for Topical Delivery of TherapeuticCompounds

One aspect of the invention provides co-polymer/enhancer formulationsfor topical delivery of therapeutics. Advantageously, theco-polymer/enhancer formulations of the present invention arethermo-sensitive, mucoadhesive or dermoadhesive, and enhance thepenetration of therapeutics across the full thickness of the skin ormucosal surfaces.

In one embodiment, the novel topical delivery formulation comprises athermo-sensitive polymer, a mucoadhesive or dermoadhesive polymer, apenetration enhancer and, optionally, one or more therapeutic agents.

In one embodiment, the co-polymer/enhancer formulation comprisesco-polymer of one or more thermosensitive polymers, one or moremucoadhesive or dermoadhesive polymers and one or morepenetration-enhancing agents. In a preferred embodiment, theco-polymer/enhancer formulation comprises co-polymer of poloxamer 188and propylene glycol, the penetration-enhancer laurocapram and,optionally, one or more therapeutic agents.

In one embodiment, the co-polymer/enhancer formulation comprises one ormore polymeric materials including, but not limited to, poloxamer andpoloxamine. Poloxamers useful according to the present inventioninclude, but are not limited to, poloxamer 188, 407, 101, 105, 108, 122,123, 124, 181, 182, 183, 184, 185, 212, 215, 217, 231, 234, 235, 237,238, 282, 284, 288, 331, 333, 334, 335, 338, 401, 402, and 403.Poloxamines useful according to the present invention include, but arenot limited to, poloxamine 304, 504, 701, 702, 704, 707, 901, 904, 908,1101, 1102, 1104, 1301, 1302, 1304, 1307, 1501, 1502, 1504, and 1508.

In certain embodiments, the co-polymer/enhancer formulation can compriseone or more polymeric materials including, but not limited to,polylactic acid and copolymers, polyvinyl acetate, celluloses andderivatives (such as carboxymethyl cellulose, cellulose acetate,cellulose acetate propionate, ethyl cellulose, hydroxypropyl methylcellulose, hydroxyalkyl methyl celluloses and alkyl celluloses),crosslinked dextrans, polyethylene glycol, diethylaminoethyl dextran,poly(cyanoacrylates), copolymers of PEG and PLA, poly(lactic-co-glycolicacid), poly(ortho esters) and hydrogels. Preferably, the polymericmaterial is pharmaceutically-acceptable, biodegradable, mucoadhesive ordermoadhesive and/or enhances the penetration of therapeutics across theskin and/or mucosal surface.

In one embodiment, the co-polymer/enhancer formulation further comprisesone or more mucoadhesive or dermoadhesive agents. In one embodiment themucoadhesive or dermoadhesive agent promotes adhesion of theco-polymer/enhancer formulation to the skin or mucosa membranes, e.g.,cervical epithelium. Preferably, the mucoadhesive or dermoadhesive agentalso enhances the penetration of therapeutics across the skin and/ormucosal surface.

Mucoadhesive or dermoadhesive agents useful according to the presentinvention include, but are not limited to, polyols such as, propyleneglycol, dipropylene glycol, polyethylene glycol, glycerine and butyleneglycol; glycol derivatives with glycerol esters, such as, oleic acidesters of propylene glycol; and non-ionizable glycol ether derivatives,such as, ethoxydiglycol.

Mucoadhesive or dermoadhesive agents useful according to the presentinvention, can also include polymers such as, polyethylene glycolcaprylic/capric glycerides; vinyl polymers (e.g., polyhydroxyethylacrylate, polyhydroxyethyl methacrylate, polyvinyl alcohol and polyvinylpyrrolidone); cellulosic derivatives, such as, methyl cellulose, ethylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose and carboxymethyl cellulose;polysaccharides, such as, alginic acid and sodium alginate.

In one embodiment, the topical delivery formulation further comprisesone or more penetration enhancers. Penetration enhancers usefulaccording to the present invention include, but are not limited to,laurocapram, diethylene glycol, monoethyl ether, n-decyl methylsulfoxide, dimethyl sulfoxide, dimethylacetamidedimethylformamide,sucrose monooleate, amides and other nitrogenous compounds (e.g., urea,2-pyrrolidone, 1-methyl-2-pyrrolidone, ethanolamine, diethanolamine andtriethanolamine), organic acids (e.g., citric acid and succinic acid),N-methyl-2-pyrrolidine, borage oil, tetrahydropiperine (THP), alcohols(e.g., methanol, ethanol, propanol, octanol, benzyl alcohol, myristylalcohol, cetyl alcohol, stearyl alcohol), fatty acids (e.g., oleicacid), fatty acid esters (e.g., isopropyl myristate, isopropylpalmitate), polyols (e.g., propylene glycol, polyethylene glycol,glycerol), polyethylene glycol monolaurate and lecithin.

In one embodiment, the penetration modifier can either enhance or retardpenetration when combined with specific mucoadhesive or dermoadhesiveagents such that, for example, it acts as a penetration enhancer incombination with propylene glycol but it acts as a penetration retardantin combination with polyethylene glycol. A co-polymer/retardantformulation may be used to prevent penetration across the skin ormucosal surfaces of harmful compounds including but not limited totoxins released during an environmental accident or catastrophe. In thisembodiment, such co-polymer/retardant formulations may serve as a formof personal protection or as a medical countermeasure (MCM) forchemical, biological, radiological, and nuclear agents, as well as thefor infectious agents, pandemic influenza and other emerging infectiousdiseases.

Preferably, the co-polymer/enhancer formulation is solid or semi-solidat room temperature and melts at a temperature slightly belowphysiological temperatures. Generally, room temperature is below 30° C.,below 28° C., below 25° C., below 23° C., below 20° C., or below 18° C.

In certain embodiments, the co-polymer/enhancer formulation melts, orbegins to melt, at a temperature ranging from about 30° C. to 42° C.,32° C. to 40° C., 33° C. to 40° C., 35° C. to 38° C., or 34° C. to 37°C. In certain embodiments, the co-polymer/enhancer formulation melts, orbegins to melt, at a temperature above 30° C., 31° C., 32° C., 33° C.,34° C., 35° C., 36° C., or 37° C. In certain embodiments, the biopolymerformulation melts, or begins to melt, at a temperature below 45° C., 44°C., 43° C., 42° C., 41° C., 40° C., 39° C., 38° C., 37° C., 36° C., 35°C., or 34° C.

The desired thermal property of the co-polymer/enhancer formulation canbe achieved by adjusting the relative ratio (e.g., in terms of weightpercentages or molar amounts) of various ingredients including, thethermo-sensitive polymeric material, the mucoadhesive agent, thepenetration enhancer and/or the therapeutic agent.

In certain embodiments, the co-polymer/enhancer formulation comprises apolymeric material at a weight percentage of about 20% to about 95%,about 25% to about 90%, about 30% to about 85%, about 35% to about 80%,about 40% to about 70%, about 50% to about 90%, about 50% to about 85%,about 60% to about 80%, about 30% to about 40%, about 30% to about 50%,about 70% to about 90%, about 70% to about 85% or about 70% to about80%.

In certain embodiments, the co-polymer/enhancer formulation comprises amucoadhesive or dermoadhesive agent at a weight percentage of about 5%to about 90%, about 10% to about 80%, about 10% to about 70%, about 10%to about 60%, about 10% to about 50%, about 10% to about 40%, about 10%to about 35%, about 10% to about 30%, about 10% to about 20%, about 5%to about 30%, about 5% to about 20%, about 5% to about 15% or about 15%to about 30%.

In certain embodiments, the co-polymer/enhancer or co-polymer/retardantformulation comprises a penetration enhancer or penetration retardant,respectively, at a concentration ranging from about 0.1 M to about 1 M,about 0.2 M to about 0.9 M, about 0.3 M to about 0.8 M, about 0.4 M toabout 0.7 M, or about 0.2 M to about 0.5 M.

In certain embodiments, the co-polymer/enhancer or co-polymer/retardantformulation comprises a penetration enhancer or penetration retardant,respectively, at a concentration above 0.05 M, 0.1 M, 0.15 M, 0.2 M, 0.3M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.5 M, 2 M, 2.5 M or3 M.

In certain embodiments, the co-polymer/enhancer or co-polymer/retardantformulation comprises a penetration enhancer or penetration retardant,respectively, at a concentration below 7 M, 6 M, 5 M, 4.5 M, 4 M, 3.5 M,3 M, 2.5 M, 2 M, 1.5 M, 1 M, 0.9 M, 0.8 M, 0.7 M, 0.6 M or 0.5 M.

In certain specific embodiments, the co-polymer/enhancer formulationcomprises poloxamer 188 and propylene glycol at a ratio (w/w) of about100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, or0:100. In preferred embodiments, the co-polymer/enhancer formulationcomprises poloxamer 188 and propylene glycol at a ratio (w/w) of about70:30 (37° C., core body temperature) or 65/35 (34° C., skintemperature).

In certain embodiments, the co-polymer/enhancer formulation compriseslaurocapram at a concentration of about 0.1 M to about 1 M, about 0.2 Mto about 0.9 M, about 0.3 M to about 0.8 M, about 0.4 M to about 0.7 Mor about 0.2 M to about 0.5 M. In a preferred embodiment, theco-polymer/enhancer formulation comprises about 0.4 M laurocapram. Theco-polymer/enhancer formulation can be used for topical delivery of avariety of small or large therapeutic agents not previously achievedusing penetration enhancers including, but not limited to, largepeptides and proteins greater than 52 amino acids, nucleic acids,compounds with unique physicochemical structures and/or properties notconsidered amenable to transdermal or transmucosal delivery,chemotherapeutic agents, anti-cancer or anti-tumor agents, antibiotics,anti-bacterial agents, anti-viral agents, anti-fungal agents,anti-microbial agents, anti-neoplastic agents, immunomodulatory agents,anti-inflammatory agents, cytokines and chemokines (e.g. interleukins),agents suitable for the treatment of diabetes such as insulinpreparations with or without secretagogues and/or thiazolidinediones,agents suitable for acute and/or chronic anti-coagulation (e.g.low-molecular weight heparin), and vaccine antigens currentlyadministered by subcutaneous or intramuscular needle injection orintravenous delivery through an indwelling catheter. In a preferredembodiment the co-polymer/enhancer formulations are useful for topicaldelivery of therapeutic agents for treatment of cervical dysplasia andfor transdermal or transmucosal delivery of large therapeuticbiomolecules such as insulin, anti-thrombotic agents and vaccineantigens.

Conversely, co-polymer/retardant formulations can be used to preventpenetration across the skin or mucosal surfaces of harmful compoundsincluding but not limited to toxins released during an environmentalaccident or catastrophe. In this embodiment, such co-polymer/retardantformulations may serve as a form of personal protection or as a medicalcountermeasure (MCM) for chemical, biological, radiological, and nuclearagents, as well as the for infectious agents, pandemic influenza andother emerging infectious diseases.

Examples of decoy peptides useful in the disclosed formulations include,but are not limited to, TRIP-Br decoy peptides including TRIP-Br1 decoypeptide (*Br1) (ATGCLLDDGLEGLFEDID) (SEQ ID NO: 1) and TRIP-Br2 decoypeptide (*Br2) (TGFLTDLTLDDILFADID) (SEQ ID NO: 2), which are describedin U.S. Pat. No. 7,223,733 (see claims 1-5).

Examples of chemotherapeutics and anti-cancer/anti-tumor agents usefulin the disclosed formulations include, but are not limited to,5-fluorouracil, chlorambucil, aminolevulinic acid, altretamine,ambomycin, vincristine, buthionine sulfoximine, asparaginase, bleomycin,busulin, trimetrexate, adriamycin, taxotere, carboplatin, cisplatin,carmustine, cladribine, 5-ethynyluracil, 9-dihydrotaxol, mitomycin,abiraterone, acivicin, teniposide, aclarubicin, acodazole hydrochloride,canarypox IL-2, acronine, thioguanine, acylfulvene, adecypenol,adozelesin, aldesleukin, thiotepa, ambamustine, busulfan, ametantroneacetate, amidox, amrubicin, mercaptopurine, cyclophosphamide,cytarabine, paclitaxel, pentostatin, dacarbazine, dactinomycin,daunorubicin, camptothecin derivatives, doxorubicin, etoposide,fludarabine phosphate, hydroxyurea, BRC/ABL antagonists, breflate,brequinar sodium, bropirimine, budotitane, amifostine, actinomycin,calcipotriol, calphostin C, calusterone, caracemide, carbetimer,floxuridine, idarubicin, ifosfamide, lomustine, mechlorethamine,melphalan, methotrexate, mitoxantrone, pliamycin, procarbazine,streptozocin and vinblastine.

In one embodiment, vaccine antigens can be, for example, tumor antigensor antigens from pathogenic organisms, such as viruses, bacteria, fungiand parasites. Thus, in some embodiments, the antigen is derived from avirus such as such but limited to, for example, hepatitis A virus (HAV),hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus(HSV), human immunodeficiency virus (HIV), human papillomavirus (HPV),cytomegalovirus (CMV), influenza virus (e.g., influenza A virus), andrabies virus. In other embodiments, the antigen is derived from abacterium such as, for example, cholera, diphtheria, tetanus,streptococcus (e.g., streptococcus A and B), Streptococcus pneumonia(e.g. over 90 serotypes, of which 88% that cause invasive disease areincluded in the 23-valent polysaccharide vaccine), pertussis, Neisseriameningitidis (e.g., meningitis A, B, C, W, Y), Neisseria gonorrhoeae,Helicobacter pylori, and Haemophilus influenza (e.g., Haemophilusinfluenza type B). In still other embodiments, thepolypeptide-containing antigen is derived from a parasite such as, forexample, a malaria parasite. Other antigens include those used toimmunize against childhood diseases, such as polio, measles, mumps andrubella.

Yet other embodiments provide for the inclusion of agents suitable forthe treatment of diabetes mellitus (types 1 and 2) and those suitablefor the prevention and treatment of thrombotic and pro-thromboticconditions (e.g. deep-vein thrombosis, pulmonary embolism, chronicatrial fibrillation, prosthetic heart valves, sickle cell). Non-limitingexamples of such agents for diabetes include insulin and insulinanalogs, (e.g., lispro (Humalog), Humulin (Isophane and Regular),Novolog (Aspart), Levemir (Detemir), Lantus (glargine) as well as othersmall molecules, such as metformin, rosiglitazone, pioglitazone andcombinations containing such molecules (e.g., metformin androsiglitazone, rosiglitazone and glimepiride). Other compounds that canbe incorporated into the disclosed composition include: biguanides, suchas metformin (Glucophage); thiazolidinediones (TZDs), such asrosiglitazone and pioglitazone; sulfonylureas, such as tolbutamide,(Orinase), acetohexamide (Dymelor), tolazamide (Tolinase),chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta,Micronase, Glynase), glimepiride (Amaryl) or gliclazide (Diamicron);Nonsulfonylurea secretagogues, such as meglitinides (e.g., repaglinide(Prandin) and nateglinide (Starlix); and alpha-glucosidase inhibitors,such as miglitol (Glyset) or acarabose (Precose, Glucobay). Non-limitingexamples of such agents for prevention or treatment of acute and/orchronic pre-thrombotic or thrombotic conditions include unfractionatedand low molecular weight heparins such as ardeparin (Normiflo),bemiparin (Hibor, Ivor, Zibor, Badyket), certoparin (Sandoparin),dalteparin (Fragmin), enoxaparin (Lovenox, Clexane), nadroparin(Fraxiparin), parnaparin (Fluxum), reviparin (Clivarin) and tinzaparin(Innohep, Logiparin); Factor Xa inhibitors such as fondaparinux(Arixtra) and idraparinux sodium (SANORG 34006, SR 34006), rivaroxaban(BAY 59-7939, Xarelto), and apixaban (Eliquis); direct thrombininhibitors such as lepirudin (Refludan), bivalirudin (Angiomax orAngiox), argatroban (Acova, Arganova, Argatra, Novastan) and dabigatran(Pradaxa, Pradax, Prazaxa); and vitamin K antagonists such as warfarin(Coumadin), Acenocoumarol (Sintrom, Sinthrome) and Phenprocoumon(Marcoumar, Marcumar, Falithrom). In certain embodiments, theco-polymer/enhancer formulation comprises a therapeutic agent at aconcentration ranging from about 0.1 mM to about 3 mM, about 0.1 mM toabout 2 mM, about 1 mM to about 1.5 mM, about 0.5 mM to about 2 mM, orabout 0.5 mM to about 1.5 mM. Amounts of therapeutic agents incorporatedinto co-polymer/enhancer formulations disclosed herein can also bedetermined by those skilled in the art (e.g., based upon age,bioavailability of a therapeutic agent, etc.) such that the therapeuticagent is delivered to a subject in amounts that effect a therapeuticbenefit to the subject.

Treatment of Cancer Via Topical Delivery of Therapeutics

Another aspect of the invention provides methods for enhanced deliveryof therapeutic agents across the skin and/or mucosal surface.Preferably, the present invention enhances topical, non-invasivedelivery of a broad range of small and large therapeutic agents ofvarious classes and compositions as well as physicochemical properties(e.g. peptides, proteins, chemicals, nucleic acids), such as but notlimited to cytotoxic decoy peptides, other chemotherapeutics andanti-tumor/anti-cancer agents, drugs for the treatment of diabetesmellitus, prevention and treatment of thrombotic and pro-thromboticconditions, and antigens for the induction of a protective immuneresponse to various vaccine antigens, as disclosed above.

In one embodiment the method comprises administering, to skin or mucosalsurface of a subject, a co-polymer/enhancer formulation of the presentinvention. In a specific embodiment the method comprises administering,to skin or mucosal surface of a subject, a co-polymer/enhancerformulation comprising poloxamer 188 and propylene glycol, laurocapramand, optionally, one or more therapeutic agents.

The term “subject,” as used herein, describes an organism, includingmammals such as primates, to which treatment with the formulationsaccording to the subject invention can be provided. Mammalian speciesthat can benefit from the disclosed methods of treatment include, butare not limited to, apes, chimpanzees, orangutans, humans, monkeys; anddomesticated animals such as dogs, cats, horses, cattle, pigs, sheep,goats, chickens, mice, rats, guinea pigs, and hamsters.

In certain embodiments the co-polymer/enhancer formulation of thepresent invention is administered to skin or mucosal surfaces including,but not limited to, cervix, vagina, anus, rectum, eye, ear, nose,thorax, vulva, larynx, and head and neck. In one embodiment theco-polymer/enhancer formulation of the present invention is topicallyadministered to cervical dysplastic lesions of the ectocervix andproximal endocervical canal of women via an intravaginal route ofmucosal delivery.

In one embodiment the present invention allows for topical delivery oftherapeutics across keratinized apical layer of skin (stratum corneum)and/or mucosa. In another embodiment the present invention allows fortopical delivery of therapeutics across non-keratinized surface of skinand/or mucosa. In one embodiment the present invention allows fortopical delivery of therapeutics into, or across, multiple layers ofcervical squamous epithelial cells. In one embodiment the presentinvention allows for topical delivery of therapeutics to the basalkeratinocytes of skin and/or mucosa.

In a specific embodiment the present invention can be used to delivertherapeutics to penetrate the keratinized surface of the mouse cervicaltransformation zone. In a specific embodiment the present invention canbe used to deliver therapeutics to penetrate non-keratinized humancervical transformation zone (T-zone) across multiple layers of squamousepithelial cells to reach the basal keratinocytes where HPV viralintegration occurs.

As exemplified in the Example, the co-polymer/enhancer formulations ofthe present invention can deliver moderate sized peptides across thekeratinized apical layer of mouse cervical T-zone. Mouse cervicaltransformation zone differs from that of human. Mouse cervix iscompletely internalized, whereas human has an ectocervix located on thevaginal surface. In addition, mouse cervix has a keratizined apicallayer in the cervical T-zone, which begins from the vagino-cervicaljunction and ends at the squamo-columnar junction. Cervical T-zone iswhere most cervical cancers arise. The mouse keratinized cervicalepithelium is comparable to keratinized skin. As exemplified in theExample, the co-polymer/enhancer formulation is capable of topicaldelivery and penetration of a 38 amino acid cytotoxic peptide across themouse keratinized cervical squamous epithelium.

In certain embodiments, the present invention can be used for topicaldelivery of therapeutics across the keratin-rich, stratum corneum ofskin to treat cutaneous neoplastic and non-neoplastic proliferativediseases including, but not limited to, HPV-associated pre-cancerous andcancerous conditions affecting vulva, vagina, anus, larynx, and head andneck, and melanoma, basal cell carcinoma, nasopharyngeal carcinomaassociated with Epstein-Barr Virus infection and psoriasis.

In one embodiment the present invention can be used for topical deliveryof chemotherapeutic and anti-cancer/anti-tumor agents for treatment oftumor or cancer including, but not limited to, human HPV-associatedcervical cancer and its precursor lesions, such as pre-cancerouslow-grade cervical dysplasia classified as cervical intraepithelialneoplasia stage I or II (CIN I, CIN II), high-grade dysplasia (CIN III),carcinoma in situ (CIS) and locally invasive or metastatic cervicalcancer (11); prostate cancer; ovarian cancer; vulvar cancer; vaginalcancer or tumor; endometrial cancer; laryngeal carcinoma; nasalpharyngeal carcinoma; bladder cancer; nasopharyngeal carcinoma, skincancer; and head and neck cancer.

In certain embodiments the present invention can be used for topical,non-invasive delivery of therapeutics for treatment of diseases orconditions, including hyperplastic skin lesions, such as, genital warts,psoriasis and keloids.

In certain embodiments the present invention can be used as anon-invasive topical transdermal or transmucosal delivery system (ordevice) applied to normal skin or mucosal surfaces to obviate the needfor subcutaneous injection of therapeutic compounds.

Formulations and Formulations for Topical Administration

The subject invention also provides for therapeutic or pharmaceuticalformulations comprising the co-polymer/enhancer formulation in a formthat can be combined with a pharmaceutically acceptable carrier. In apreferred embodiment the therapeutic or pharmaceutical formulation issolid or semi-solid at room temperature and melts at a temperatureslightly below desired physiological temperatures.

The term “carrier” refers to a diluent, adjuvant, excipient or vehiclewith which the compound is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum oil such as mineral oil, vegetable oil such as peanut oil,soybean oil and sesame oil, animal oil or oil of synthetic origin.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene glycol, water, ethanol and the like. The therapeuticformulation, if desired, can also contain minor amounts of wetting,emulsifying or pH buffering agents. These formulations can take the formof creams, foam, patches, lotions, drops, sprays, gel, oils, aerosol,powders, ointment, solutions, suspensions, emulsion and the like. Theformulation can be formulated with traditional binders and carriers suchas triglycerides. Examples of suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin. Suchformulations contain a therapeutically effective amount of thetherapeutic formulation, together with a suitable amount of carrier soas to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

The subject invention also provides for the modification of theingredient such that it is more stable once administered to a subject,i.e., once administered it has a longer time period of effectiveness ascompared to the unmodified form. Such modifications are well known tothose of skill in the art, e.g., microencapsulation, etc.

The amount of the therapeutic or pharmaceutical formulation of theinvention which is effective in the treatment of a particular disease,condition or disorder will depend on the route of administration and theseriousness of the disease, condition or disorder and should be decidedaccording to the judgment of the practitioner and each patient'scircumstances.

Further, the present invention provides kits containing therapeuticagents such as, lyophilized cytotoxic decoy peptides, vehicle and/orco-polymer/enhancer formulations. Preferably, the formulations of thepresent invention are stable in a wide range of temperatures below thedesired melting temperature. In one embodiment the active therapeuticagents can be reconstituted by mixing pre-measured quantities of eachcomponent immediately prior to use.

EXAMPLES

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 Thermo-Sensitive, Mucoadhesive, and Penetration-EnhancingFormulations

To create co-polymer/enhancer formulations with desired thermo-sensitiveproperties, USP grade poloxamer 188 (P188) and propylene glycol (PG)were mixed at various ratios (100/0, 80/20, 70/30, 50/50 and 0/100) andheated to 60° C. The co-polymer/enhancer preparations were suctionedinto pre-warmed (60° C.) Silastic tubing (⅛^(th)-inch inner diameter;Fisher, Pittsburgh, Pa.), solidified at room temperature and extrudedfrom the tube with compressed air (12). 3 mm samples were cut from thesolid “rope” with a scalpel.

The thermal profile of the P188/PG co-polymer preparations wasdetermined using a DSC 6200/Exstar 6000 (Seiko Instruments) DifferentialScanning calorimeter. To obtain co-polymer preparations that have thedesired melting point (30-37° C.) in the presence of 1 mM of variousdecoy peptides, DSC 6200 was programmed with 3 cycles of heating at 10°C./min and cooling at 50° C./min, with 2 min equilibration times betweenheating/cooling cycles. Measurements were taken at a temperature rangeof −25-65° C.

The DSC curves did not exhibit dual peaks, indicating that the P188/PGco-polymer preparations were in a homogeneous phase. The co-polymer withan 80/20 ratio of P188/PG exhibited the desired thermal property, thatis, solid at room temperature and instantly melted at a temperatureslightly below the physiological temperature of 37° C.

The P188/PG (80/20 ratio) co-polymer was then used to deliverFITC-tagged 38 amino acid cytotoxic decoy peptides. The P188/PGco-polymer (80/20 ratio) alone was unable to penetrate the keratinizedapical layer of the mouse cervix, as determined by direct visualizationusing fluorescence microscopy (data not shown).

Laurocapram is a non-toxic enhancer of cutaneous penetration (13, 14).It is among a class of “penetration modifiers” (15, 16) that, dependingon the vehicle formulation, can enhance or retard the penetration ofhuman stratum corneum. DSC and spectral analysis revealed thatpenetration modifier formulations (e.g., laurocapram) disrupt andfluidize the stratum corneum lipid bilayers (17). Since 1984,laurocapram has been widely used as a safe penetration enhancer and hasbeen formulated with propylene glycol—a mucoadhesive and dermoadhesiveagent (17, 18).

0.4 M laurocapram (17) was added to the P188/PG preparations. The DSCcurves (FIG. 1) show that the formulation composed of 1 mM of thecytotoxic decoy peptide *Br1, a 70/30 ratio of P188/PG co-polymer and0.4 M laurocapram has the desired thermo-sensitive properties fordelivery of cytotoxic decoy peptides (delivery of *Br2 is not shown). Asshown below, this new formulation can enhance penetration of cytotoxicdecoy peptides across the keratinized surface of mouse cervical mucosa,which resembles the stratum corneum of skin.

The entire mouse cervical T-zone begins from the cervico-vaginaljunction to the termination of the cervical T-zone, which isapproximately one-third of the distance into one of the 2cervical/uterine horns. To achieve topical delivery of decoy peptidesinto the entire mouse cervical T-zone, a solid FITC-tagged decoypeptide-containing co-polymer/enhancer rod was manually delivered intothe endocervical canal and into one of the 2 cervical/uterine horns of a7 month old K14E6 homozygous female transgenic mouse expressing the HPVtype 16 E6 oncoprotein that synergizes with 17β-estradiol to inducelow-grade dysplasia (CIN I, II) in situ that closely mimics thestep-wise development of human cervical cancer; the “untreated” cervicalserved as an internal control.

After animals were anesthetized, a “speculum” comprised of a standardP-100 pipette tip with the tip cut off at the midpoint along its lengthwas inserted into the vagina to expose the opening of the cervicalcanal. A three-fourth inch long, 24-gauge catheter (Teruma Surflush;inner diameter 0.47 mm) with the trocar removed was fit onto acompatible P-10 micropipette. The catheter was loaded with thepeptide-containing, liquid co-polymer/enhancer formulation to a lengththat approximates the entire cervical T-zone and extends across thesquamo-columnar junction into the uterine horn.

After the co-polymer/enhancer formulation solidified into rod shape atroom temperature, the tip of the catheter was inserted into the entranceof the cervix, and the solid rod was pushed into the cervical canal bythe trocar; meanwhile, the catheter tip was withdrawn so that, at theend of the procedure, the rod protruded ˜2 mm outside of the cervicalcanal. It is observed that the co-polymer/enhancer formulation underwentphase transition from a solid rod into a consistent gel-likeformulation, without leakage from the cervical opening.

Six hours after peptide delivery the animals were euthanized. The entirereproductive tract was surgically removed, fixed with formalin andembedded for histological sectioning as described (19).

1 mM FITC-conjugated *Br1 was used for direct visualization of peptidedelivery by fluorescence microscopy. Fluorescence microscopy showed thatboth formulations, which contain P188 and PG, are mucoadhesive to thekeratinized apical surface.

FIG. 2 shows a direct comparison between the penetration property of thelauracopram-containing and the non-lauracopram-containing formulation ina single mouse reproductive tract. A rod consisting of the 80/20 P188/PGformulation (without lauracopram) was introduced high up into the rightcervical/uterine horn under ultrasound guidance, while a rod consistingof the 70/30 formulation and 0.4M lauracopram was introduced into theleft cervical/uterine horn.

Specifically, penetration across the apical keratin layer of cervicalsquamous epithelium was only observed in the left cervical/uterine horn,where the rod contained 0.4 M lauracopram (FIG. 2B, 2B′). It is evidentthat the lauracopram-containing rod penetrated through multiple layersof cervical squamous epithelial cells, when comparing fluorescencemicroscopic imaging of an unstained section (FIG. 2B) with lightmicroscopic examination of a consecutive H&E stained section (FIG. 2A).

All patents, patent applications, provisional applications andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

REFERENCES

-   1. Garcia M, Jemal, A., Ward, E. M., Center, M. M., Hao, Y.,    Siegel, R. L., and Thun, M. J. 2008. Global Cancer Facts & FIGS.    2007. Atlanta, Ga.: American Cancer Society-   4. de Sanjose S et al., Human papillomavirus genotype attribution in    invasive cervical cancer: a retrospective cross-sectional worldwide    study. Lancet Oncol 11: 1048-56-   5. Munoz N, Bosch F X, de Sanjose S, Herrero R, Castellsague X, Shah    K V, Snijders P J, Meijer C J. 2003. Epidemiologic classification of    human papillomavirus types associated with cervical cancer. N Engl J    Med 348: 518-27-   6. Walboomers J M, Jacobs M V, Manos M M, Bosch F X, Kummer J A,    Shah K V, Snijders P J, Peto J, Meijer C J, Munoz N. 1999. Human    papillomavirus is a necessary cause of invasive cervical cancer    worldwide. J Pathol 189: 12-9-   8. Hsu S I, Yang C M, Sim K G, Hentschel D M, O'Leary E, Bonventre    J V. 2001. TRIP-Br: a novel family of PHD zinc finger- and    bromodomain-interacting proteins that regulate the transcriptional    activity of E2F-1/DP-1. EMBO J 20: 2273-85-   9. Sim K G, Zang Z, Yang C M, Bonventre J V, Hsu S I. 2004. TRIP-Br    links E2F to novel functions in the regulation of cyclin E    expression during cell cycle progression and in the maintenance of    genomic stability. Cell Cycle 3: 1296-304-   10. Zang Z J, Sim K G, Cheong J K, Yang C M, Yap C S, Hsu S I. 2007.    Exploiting the TRIP-Br Family of Cell Cycle Regulatory Proteins as    Chemotherapeutic Drug Targets in Human Cancer. Cancer Biol Ther 6-   11. DeMay M. 2007. Practical principles of cytopathology (Revised    edition). Chicago: American Society for Clinical Pathology Press-   13. Stoughton R B. 1982. Enhanced percutaneous penetration with    1-dodecylazacycloheptan-2-one. Arch Dermatol 118: 474-7-   14. Stoughton R B, and McClure, W. O. 1983. Azone; a new non-toxic    enhancer of cutaneous penetration. Drug Development and Industrial    Pharmacy 9: 725-44-   15. Kaushik D, Batheja P, Kilfoyle B, Rai V, Michniak-Kohn B. 2008.    Percutaneous permeation modifiers: enhancement versus retardation.    Expert Opin Drug Deliv 5: 517-29-   16. Kaushik D, Costache A, Michniak-Kohn B. 2010. Percutaneous    penetration modifiers and formulation effects. Int J Pharm 386:    42-51-   17. Kaushik D, Michniak-Kohn B. 2010. Percutaneous penetration    modifiers and formulation effects: thermal and spectral analyses.    AAPS Pharm Sci Tech 11: 1068-83-   18. Spruance S L, McKeough M, Sugibayashi K, Robertson F, Gaede P,    Clark D S. 1984. Effect of azone and propylene glycol on penetration    of trifluorothymidine through skin and efficacy of different topical    formulations against cutaneous herpes simplex virus infections in    guinea pigs. Antimicrob Agents Chemother 26: 819-23-   19. Riley R R, Duensing S, Brake T, Munger K, Lambert P F, Arbeit    J M. 2003. Dissection of human papillomavirus E6 and E7 function in    transgenic mouse models of cervical carcinogenesis. Cancer Res 63:    4862-71

What is claimed is:
 1. A co-polymer/enhancer formulation for topicaldelivery of therapeutics across the skin or mucosal surfaces, comprisinga thermosensitive polymer, a mucoadhesive or dermoadhesive polymer, apenetration enhancer and a therapeutic agent.
 2. The co-polymer/enhancerformulation according to claim 1, wherein said formulation comprisespoloxamer 188, propylene glycol, laurocapram and a therapeutic agent. 3.The co-polymer/enhancer formulation according to claim 1, wherein thetherapeutic agent is selected from decoy peptides, chemotherapeuticagents, anti-cancer or anti-tumor agents, antibiotics, anti-bacterialagents, anti-viral agents, anti-fungal agents, anti-microbial agents,anti-neoplastic agents, immunomodulatory agents, anti-inflammatoryagents, cytokines, chemokines or interleukins, nucleic acids and avaccine antigen is selected from the group consisting of: tumorantigens, viral antigens, bacterial antigens, fungal antigens, parasiteantigens and allergens.
 4. The co-polymer/enhancer formulation accordingto claim 1, wherein the therapeutic agent is wherein the therapeuticagent is insulin, an insulin analog, a biguanide, a thiazolidinedione, asulfonylurea, a nonsulfonylurea secretagogue, an alpha-glucosidaseinhibitor or combinations thereof.
 5. The co-polymer/enhancerformulation according to claim 4, wherein said insulin analog is lispro(Humalog), Humulin (Isophane and Regular), Novolog (Aspart), Levemir(Detemir), or Lantus (glargine).
 6. The co-polymer/enhancer formulationaccording to claim 1, wherein the therapeutic agent is metformin,rosiglitazone, pioglitazone, troglitazone, tolbutamide, acetohexamide,tolazamide, chlorpropamide, glipizide, glyburide, glimepiride,gliclazide, repaglinide, nateglinide miglitol, acarabose or combinationsthereof.
 7. The co-polymer/enhancer formulation according to claim 1,wherein said therapeutic agent is an anti-thrombotic administered toprevent or treat pro-thrombotic or thrombotic conditions.
 8. A method oftreating diabetes comprising the topical or mucosal administration of aco-polymer/enhancer formulation according to claim 4 to a subject havingdiabetes in an amount effective to treat diabetes.
 9. A method ofinducing an immune response in a subject comprising the topical ormucosal administration of a co-polymer/enhancer formulation according toclaim 3 to a subject in an amount effective to induce an immune responseto an vaccine antigen contained within said formulation, wherein saidvaccine antigen is selected from the group consisting of: tumorantigens, viral antigens, bacterial antigens, fungal antigens, parasiteantigens and allergens.
 10. A method of treating pro-thrombotic orthrombotic conditions comprising the topical or mucosal administrationof a co-polymer/enhancer formulation according to claim 7 in an amounteffective to prevent or treat a pro-thrombotic or thrombotic condition.